Integrity of the blood‐brain barrier and changes in the microvasculature during the progression of Alzheimer’s disease in the 3XTG‐AD model

Abstract

AbstractBackgroundThere is evidence that vasculature failures are related with the development of Alzheimer´s. Between 60 and 90% of patients with AD present with pathologies of cerebrovascular disease. The blood brain barrier (BBB) is a highly selective, semipermeable chemical and structural barrier that ensures a stable internal environment in the brain. BBB dysfunction was initially identified in animal models for AD, and confirmed in patients. To date it is not clear how it originates. Physically, BBB endothelial cells are bound by tight junction complexes (TJ), formed by plasma membrane proteins, such as ocludins and claudins and cytoplasmic scaffolding proteins, such as ZO1. The study of changes in the expression of TJ proteins in a 3xTg‐AD mouse model at different ages will allow us to determine if damage to the BBB occurs before the appearance of amyloid‐β plaques.MethodThe animal's brain (3xTg‐AD mice of 9 and 13 months) is removed. Subsequently, the microvasculature is precipitated by density‐dependent centrifugation through the use of a solution with dextran, managing to separate the cerebral parenchyma and the myelin from the microvasculature. Once the microvessels are obtained, we incubate with anti‐ZO‐1, claudin‐5 and ocludin fluorescent primary antibodies and secondary antibodies (Alexas 488 and 546) and Hoechst 33342. Isolated microvessels are mixed with a fluorescent mounting medium (Fluoromount), mounted on slides and covered with a coverslip. Once mounted, they are scanned with a confocal laser scanning microscope.ResultWe found a decrease in the expression of tight junction proteins in 3xTg‐AD mice of 13 months compared to the same model but at 9 months and also compared to control animals. Morphological differences were also observed in the microvessels of the animals 3xTg‐AD in comparison with the WT and between the animals of 9 and 13 months, with morphological damage being more evident in the older animals.ConclusionThis could be telling us that the BBB is compromised. We still have to analyze the association between this decrease in TJ proteins and the accumulation of beta‐amyloid, in addition to analyzing it at earlier ages in order to establish from what age these changes in this murine model are measurable.